Intra-vital microscopy using non-linear optical techniques

这些研究的目的是发展成像技术来监测亚细胞结构和过程，在体内。使用的主要方法是非线性光学显微镜技术。我们一直在系统地开发一种适应生物组织和结构的体内光学显微镜系统，而不是强迫动物在传统的显微镜台上。在过去的一年中取得了以下主要发现：1)微创，双光子激发荧光显微镜（TPEFM）被用于研究细胞内的亚细胞代谢过程，在完整的动物，在正常的体内条件下，使用各种外源性和内在荧光探针。我们修改了我们的运动跟踪方案，以执行全三维运动跟踪，以补偿所有三个维度的生理运动。利用近实时计算机中的图形处理单元和显微镜中的共振扫描镜，我们能够跟踪250毫秒左右的组织运动。这允许在体内在微米尺度上对细胞内的大多数缓慢运动进行校正。2)我们已经扩展了我们的系统，允许混合双光子和单光子激发方案，允许使用近紫外激光在体内组织内进行光转换探针。我们对这种适应的主要目标是在转基因小鼠系中使用一种针对线粒体的光转化蛋白来跟踪线粒体运动和融合事件。3)我们扩大了在体内血管床结构测定方面的努力。使用最先进的图像处理方法，我们已经能够以如此高的保真度分割出组织内的血管结构，我们可以从这些数据中预测二氧化硅中的血管流动模式。这为各种组织的微循环的结构和控制提供了新的见解，包括单个肌肉群内血管流的慢速和快速收缩肌纤维的分割。3)在这些研究中，我们一直在继续研究使用自适应光学来校正激发光的畸变。使用系统的点扩展函数的新方法以及迭代图像分析方法开始对图像进行重要的一阶校正，我们希望在下一个报告期内提高图像深度，分辨率和功率要求。4)我们一直与商业伙伴合作开发一套全辐射探测系统，以改善TPEFM的信噪比和效率。正如在之前的报告中所讨论的那样，该系统收集组织发出的所有散射光来创建图像，而不是使用显微镜物镜收集的光。这使得传统检测系统的信噪比提高了3到4倍。商业样机将于今年秋天在实验室安装。

The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. The major approach used was non-linear optical microscopy techniques. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. The following major findings were made over the last year: 1) Minimally invasive, two photon excitation fluorescence microscopy (TPEFM) is being used to study sub-cellular metabolic processes within cells, in intact animals, under normal in vivo conditions using various exogenous and intrinsic fluorescent probes. We have modified our motion tracking schemes to perform full three dimensional motion tracking to compensate for physiological motion in all three dimensions. Using a graphical processing units in a near real time computer and a resonant scanning mirror in the microscope, we able to track tissue motion on the order of 250 msec. This permits the correction of most slow motions inside cells on the micron scale, in vivo. 2) We have expanded our system to permit hybrid two and one photon excitation schemes to permit the use of near UV lasers to photoconvert probes within the tissues in vivo. Our primary target for this adaptation is the tracking mitochondrial motion and fusion events using a photoconverting protein genetically targeted to mitochondria in a transgenic mouse line. 3) We have expanded our efforts in vascular bed structure determination in vivo. Using state of the art image processing approaches we have been able to segment out the vascular structures inside tissues at such a high fidelity that we can predict vascular flow patterns in silica from these data. This is providing new insights into the structure and control of the microcirculation of various tissues, including the segmentation of vascular flow to slow and fast twitch muscle fibers within a single muscle group. 3) We have been continuing our studies on the use of adaptive optics to correct for the distortion of the excitation light in these studies. New methods of using the point spread function of the system as well as iterative image analysis approaches are beginning to make significant first order corrections to the images that we hope will improve image depth, resolution and power requirements in the next reporting period. 4) We have been working with a commercial partner on the development of a total emission detection system to improve the signal to noise and efficiency of TPEFM. This system, as discussed in previous reports, collects all of the scattered light emitting from a tissue to create the image rather than using the light collected by the microscope objective. This results in an improvement of a factor of 3 or 4 in signal to noise from conventional detection systems. The commercial prototype is being installed in the laboratory this fall.